Next-Generation Sequencing–Based Diagnosis of Unexplained Inherited Hemolytic Anemias Reveals Wide Genetic and Phenotypic Heterogeneity
2020; Elsevier BV; Volume: 22; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2020.01.007
ISSN1943-7811
AutoresManu Jamwal, Anu Aggarwal, Arindam Palodhi, Prashant Sharma, Deepak Bansal, Amita Trehan, Pankaj Malhotra, Arindam Maitra, Reena Das,
Tópico(s)Blood groups and transfusion
ResumoDetermination of the cause of inherited hemolysis is based on clinical and stepwise conventional laboratory tests. Patients with obscure etiology require genetic diagnosis, which is time-consuming, expensive, and laborious, mainly because of numerous causal genes. This study enrolled 43 patients with clinical and laboratory evidence of unexplained hemolytic anemia. Initially, 13 patients were tested using a commercial (TruSight One) panel, and remaining cases underwent targeted sequencing using a customized 55-gene panel. Pyruvate kinase deficiency was found in eight, glucose-6-phosphate dehydrogenase (G6PD) deficiency in three (G6PD Guadalajara in two and p.Tyr227Ser: novel, named as G6PD Chandigarh), and glucose-6-phosphate isomerase (GPI) deficiency in two (GPI:p.Arg347His and p.Phe304Leu: novel, named as GPI Chandigarh). Three patients had Mediterranean stomatocytosis/macrothrombocytopenia, and two had overhydrated stomatocytosis. Xerocytosis was found in three patients, whereas six had potentially pathogenic variants in membrane protein–coding genes. Overall, 63% cases received a definite diagnosis. Timely determination of etiology was helpful in diagnosis, genetic counseling, and offering a prenatal diagnosis. Therapeutic implications include performing or avoiding splenectomy that may ameliorate the anemia in many but also predispose to thrombosis in other groups of patients. This first study on the genetic spectrum of unexplained hemolytic anemia from the Indian subcontinent also represents, currently, one of the largest cohort worldwide of such patients. Determination of the cause of inherited hemolysis is based on clinical and stepwise conventional laboratory tests. Patients with obscure etiology require genetic diagnosis, which is time-consuming, expensive, and laborious, mainly because of numerous causal genes. This study enrolled 43 patients with clinical and laboratory evidence of unexplained hemolytic anemia. Initially, 13 patients were tested using a commercial (TruSight One) panel, and remaining cases underwent targeted sequencing using a customized 55-gene panel. Pyruvate kinase deficiency was found in eight, glucose-6-phosphate dehydrogenase (G6PD) deficiency in three (G6PD Guadalajara in two and p.Tyr227Ser: novel, named as G6PD Chandigarh), and glucose-6-phosphate isomerase (GPI) deficiency in two (GPI:p.Arg347His and p.Phe304Leu: novel, named as GPI Chandigarh). Three patients had Mediterranean stomatocytosis/macrothrombocytopenia, and two had overhydrated stomatocytosis. Xerocytosis was found in three patients, whereas six had potentially pathogenic variants in membrane protein–coding genes. Overall, 63% cases received a definite diagnosis. Timely determination of etiology was helpful in diagnosis, genetic counseling, and offering a prenatal diagnosis. Therapeutic implications include performing or avoiding splenectomy that may ameliorate the anemia in many but also predispose to thrombosis in other groups of patients. This first study on the genetic spectrum of unexplained hemolytic anemia from the Indian subcontinent also represents, currently, one of the largest cohort worldwide of such patients. Anemia is the most common blood disorder, affecting >1.5 billion individuals worldwide.1McLean E. Cogswell M. Egli I. Wojdyla D. De Benoist B. Worldwide prevalence of anaemia, WHO Vitamin and Mineral Nutrition Information System, 1993-2005.Public Health Nutr. 2009; 12: 444-454Crossref PubMed Scopus (1365) Google Scholar Leading causes include nutritional deficiencies, blood loss, ineffective hematopoiesis (including inflammation, malignancies, and infections), and hemolysis. Patients with hemolytic anemias display increased red cell destruction, leading to absolute reticulocytosis with elevated unconjugated bilirubin and lactate dehydrogenase levels, episodic jaundice, and splenomegaly. Phenotypes vary from severe transfusion-dependent anemia (eg, β-thalassemia major) to fully compensated hemolysis [eg, mild hereditary spherocytosis (HS)]. Those with severe inherited hemolysis tend to present early in life and often have distinctive clinical/hematological features that correlate with the underlying genetic etiology. β-Thalassemia and α-thalassemia, symptomatic hemoglobinopathies, and glucose-6-phosphate dehydrogenase (G6PD) deficiency are common causes of inherited hemolysis in South-East Asia, whereas pyruvate kinase (PK) deficiency, uncommon or unstable hemoglobin variants, and membranopathies are encountered less commonly. A stepwise diagnostic algorithm is usually applied in inherited anemias. However, the etiology in a significant subset of patients with definite clinical and laboratory evidence of hemolysis remains unexplained, even after completion of routine testing.2Roy N.B.A. Wilson E.A. Henderson S. Wray K. Babbs C. Okoli S. Atoyebi W. Mixon A. Cahill M.R. Carey P. Cullis J. Curtin J. Dreau H. Ferguson D.J.P. Gibson B. Hall G. Mason J. Morgan M. Proven M. Qureshi A. Sanchez Garcia J. Sirachainan N. Teo J. Tedgård U. Higgs D. Roberts D. Roberts I. Schuh A. A novel 33-gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.Br J Haematol. 2016; 175: 318-330Crossref PubMed Scopus (56) Google Scholar,3Agarwal A.M. Nussenzveig R.H. Reading N.S. Patel J.L. Sangle N. Salama M.E. Prchal J.T. Perkins S.L. Yaish H.M. Christensen R.D. Clinical utility of next-generation sequencing in the diagnosis of hereditary haemolytic anaemias.Br J Haematol. 2016; 174: 806-814Crossref PubMed Scopus (60) Google Scholar Molecular diagnosis establishes the precise etiology, aids evidence-based prognostication, and provides a potential tool for prenatal diagnosis in the family. However, genetic studies based on Sanger sequencing are often not feasible because of the numerous potentially causal genes and their frequent large sizes and complexities (Figure 1). In recent years, massively parallel, targeted next-generation sequencing (NGS) of candidate genes has expedited molecular diagnosis in such disorders. The success rate of NGS in unexplained hemolytic anemias has ranged from 39% to 70%.2Roy N.B.A. Wilson E.A. Henderson S. Wray K. Babbs C. Okoli S. Atoyebi W. Mixon A. Cahill M.R. Carey P. Cullis J. Curtin J. Dreau H. Ferguson D.J.P. Gibson B. Hall G. Mason J. Morgan M. Proven M. Qureshi A. Sanchez Garcia J. Sirachainan N. Teo J. Tedgård U. Higgs D. Roberts D. Roberts I. Schuh A. A novel 33-gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.Br J Haematol. 2016; 175: 318-330Crossref PubMed Scopus (56) Google Scholar, 3Agarwal A.M. Nussenzveig R.H. Reading N.S. Patel J.L. Sangle N. Salama M.E. Prchal J.T. Perkins S.L. Yaish H.M. Christensen R.D. Clinical utility of next-generation sequencing in the diagnosis of hereditary haemolytic anaemias.Br J Haematol. 2016; 174: 806-814Crossref PubMed Scopus (60) Google Scholar, 4Shefer Averbuch N. Steinberg-Shemer O. Dgany O. Krasnov T. Noy-Lotan S. Yacobovich J. Kuperman A.A. Kattamis A. Ben Barak A. Roth-Jelinek B. Chubar E. Shabad E. Dufort G. Ellis M. Wolach O. Pazgal I. Abu Quider A. Miskin H. Tamary H. Targeted next generation sequencing for the diagnosis of patients with rare congenital anemias.Eur J Haematol. 2018; 101: 297-304Crossref PubMed Scopus (24) Google Scholar Many patients in India lack access to the relatively expensive and technically complex NGS facilities. The current study reports the largest worldwide cohort of unexplained inherited hemolysis tested using NGS. This first systematic study from the Indian subcontinent reveals wide clinical and molecular heterogeneity among the patients. The study was conducted over a 5-year period in a tertiary care hospital and research institute in northern India. It was approved by the Institutional Ethics Committee vide PGI/IEC/2015/837. Patients were enrolled after informed consent or, in the case of minors, assent from the patient and consent from the legal guardian(s). All provisions of the Declaration of Helsinki and its later amendments were followed. Patients with clinical and/or laboratory evidence of hemolytic anemia that remained unexplained after extended routine investigations were enrolled. Likely inherited hemolytic anemia was considered in view of factors like early onset in life, positive family history, requirement for blood transfusion(s), splenomegaly, recurrent jaundice with unconjugated hyperbilirubinemia (>0.7 mg/dL), stigmata of chronic anemia/hemolysis for growth failure, hemolytic facies, hypogonadism, pigment gallstones at age 2.5% after correction), elevated plasma hemoglobin (>15 mg/dL), and elevated lactate dehydrogenase (>250 U/L). Patients with any combination of the above features showing compensated hemolysis (as suggested by a normal range hemoglobin value for age and sex accompanied by reticulocytosis) were also enrolled. G6PD deficiency, hemoglobinopathies (thalassemia syndromes, sickle cell disorders, and hemoglobin E syndromes), immune hemolysis, unstable hemoglobins, PK deficiency, and HS were excluded (details below). Bone marrow was examined if indicated, usually to exclude congenital dyserythropoietic anemia or myelodysplasia. Approximately 3 to 5 mL peripheral blood was collected in K2EDTA from the patient as well as affected and unaffected parents and siblings (wherever possible) to establish pathogenicity of the molecular variant and inheritance patterns. Complete blood counts, including reticulocyte count, were performed on automated hematology analyzers (LH780; Beckman Coulter, Brea, CA). Manual reticulocyte counts were performed by incubating erythrocytes with new methylene blue stain (1:1 dilution; 20 minutes). Methylene blue reduction test was performed for G6PD-deficiency screening. Hemoglobin high-performance liquid chromatography (Variant II analyzer; Bio-Rad Laboratories, Hercules, CA) and hemoglobin electrophoresis (GenioS automated instrument; Interlab, Rome, Italy) were performed for hemoglobinopathies and thalassemia syndromes. Autoimmune hemolysis was excluded by direct antiglobulin test. Unstable hemoglobins were screened by Heinz bodies' demonstration and the heat and isopropanol stability tests. Flow cytometry–based eosin 5′ maleimide (EMA) dye–binding test was performed, as previously described,5Joshi P. Aggarwal A. Jamwal M. Sachdeva M.U.S.S. Bansal D. Malhotra P. Sharma P. Das R. A comparative evaluation of eosin-5′-maleimide flow cytometry reveals a high diagnostic efficacy for hereditary spherocytosis.Int J Lab Hematol. 2016; 38: 520-526Crossref PubMed Scopus (17) Google Scholar along with incubated osmotic fragility testing to diagnose HS. Red cell PK activity was measured spectrophotometrically.6Kedar P. Hamada T. Warang P. Nadkarni A. Shimizu K. Fujji H. Ghosh K. Kanno H. Colah R. Spectrum of novel mutations in the human PKLR gene in pyruvate kinase-deficient Indian patients with heterogeneous clinical phenotypes.Clin Genet. 2009; 75: 157-162Crossref PubMed Scopus (11) Google Scholar Because the genetic defects expected in Indian patients with unexplained hemolytic anemia were previously unstudied, the initial 13 unrelated patients were tested with a commercially available, comprehensive TruSight One sequencing panel that covered 4813 clinically relevant genes (Illumina, San Diego, CA). Genomic DNA was extracted with QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) and quantified spectrophotometrically using NanoDrop 2000 (ThermoFisher Scientific Inc., Waltham, MA) and by PicoGreen assay in Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA). Libraries were prepared using 50 ng of DNA that was tagmented, amplified, hybridized with the probes, and captured using biotin-streptavidin chemistry. Libraries were recaptured and amplified to get highly specific enrichment. Their quality was assessed on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and by PicoGreen assay in Qubit 2.0 Fluorometer. The enriched DNA libraries (10 pmol) were spiked with 1% PhiX libraries, and sequencing was performed on the Illumina MiSeq sequencing system using 300 cycle (150 × 2) chemistry. MiSeq Reporter and VariantStudio version 2.1 software (both from Illumina) were used for analysis, classification, and reporting of genomic variants. The .vcf files generated by MiSeq Reporter were imported to VariantStudio for annotation and variant prioritization. Sorting Intolerant from Tolerant (SIFT) and PolyPhen results were integrated in the VariantStudio pipeline. Because of the vast number of variants detected, in silico screening of variants that might represent probably causal variants was performed in decreasing order of likelihood, as suggested by the clinical and laboratory features of that particular patient/family. The 4813 genes sequenced by TruSight One Sequencing Panel were narrowed down during bioinformatics data analysis based on, to begin with, the likely implicated genes suggested by the clinical phenotype. If no causal variant was discovered in them, the remaining genes as short listed by literature search (Online Mendelian Inheritance in Man and PubMed) were analyzed. Integrative Genome Viewer was used for visualizing sequence data.7Robinson J.T. Thorvaldsdóttir H. Winckler W. Guttman M. Lander E.S. Getz G. Mesirov J.P. Integrative genomics viewer.Nat Biotechnol. 2011; 29: 24-26Crossref PubMed Scopus (7655) Google Scholar All significant variants were confirmed/excluded by Sanger sequencing of either symptomatic or asymptomatic parents (as available) and siblings (affected sibling available in three cases). Because of the high cost of the TruSight One Sequencing Panel, testing was streamlined using the data obtained from the 13 cases to design a customized, more focused gene panel. Online Mendelian Inheritance in Man and literature were searched exhaustively to finalize a list of 55 genes implicated in inherited anemias to be targeted for sequencing (Figure 1 and Supplemental Table S1). Primers were designed using the web-based DesignStudio tool (Illumina) covering coding regions, 5′ and 3′ untranslated regions, and promoters of the candidate genes. Finally, the custom panel had 1525 amplicon regions with a total size of 360,381 bp. A further 30 unrelated patients were enrolled after excluding common causes of hemolysis and tested with this TruSeq Custom Amplicon panel (Illumina). A targeted pull-down and amplification assay was developed for the selected genes using TruSeq Custom Amplicon version 1.5 (Illumina). DNA libraries were prepared using Illumina's TruSeq version 1.5 customized panel, as per the manufacturer's instructions. Briefly, 250 ng of input DNA was tagmented and barcoded, and samples were pooled to a single pull down. The quality and quantity of these libraries were assessed in Bioanalyzer 2100 by High Sensitivity DNA kit and by PicoGreen assay in Qubit 2.0 fluorometer. Library pools (6 pmol) were sequenced in a rapid run mode in HiSeq 2500 (Illumina) platform with 150 × 2 bp paired-end reads. Sequence data were exported to BaseSpace and analyzed using the automated TruSeq Amplicon pipeline. The .vcf file generated from analyzed data was downloaded and imported to the VariantStudio software for annotation and further analysis. As there is a possibility of missing the variants in automated pipelines as a result of genetic isoform complexity,8Jamwal M. Aggarwal A. Palodi A. Sharma P. Bansal D. Maitra A. Das R. A nonsense variant in the hexokinase 1 gene (HK1) causing severe non-spherocytic haemolytic anaemia: genetic analysis exemplifies ambiguity due to multiple Isoforms.Br J Haematol. 2019; 186: e142-e145Crossref PubMed Scopus (6) Google Scholar a parallel data analysis using BWA-MEM9Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM.ArXiv Prepr ArXiv. 2013; (1303.3997v1 (q-bio.GN))Google Scholar and Varscan210Koboldt D.C. Zhang Q. Larson D.E. Shen D. McLellan M.D. Lin L. Miller C.A. Mardis E.R. Ding L. Wilson R.K. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing.Genome Res. 2012; 22: 568-576Crossref PubMed Scopus (2963) Google Scholar was also performed. All the potentially pathogenic variants were detected in both methods. All nonsynonymous amino acid variants were analyzed by Protein Variation Effect Analyzer (PROVEAN), MutPred, Mendelian Clinically Applicable Pathogenicity, and Combined Annotation Dependent Depletion to assess any potentially damaging effect/s. Splice site variants were analyzed by Human Splicing Finder (http://www.umd.be/HSF3/HSF.shtml, last accessed July 25, 2019). Variants passing these filtering steps were considered to be most likely disease causing and were validated by Sanger sequencing (ABI 3130 Genetic Analyzer; Applied Biosystems, Waltham, MA) in the patients as well as their affected (available in two cases)/unaffected family members. A previously described testing algorithm was used in the diagnosis of inherited hemolytic anemias.11Jamwal M. Aggarwal A. Das A. Maitra A. Sharma P. Krishnan S. Arora N. Bansal D. Das R. Next-generation sequencing unravels homozygous mutation in glucose-6-phosphate isomerase, GPIc.1040G>A (p.Arg347His) causing hemolysis in an Indian infant.Clin Chim Acta. 2017; 468: 81-84Crossref PubMed Scopus (15) Google Scholar After careful consideration, 43 unrelated subjects fulfilling the diagnostic criteria were enrolled, including 28 (65.1%) males and 15 (34.9%) females. Age at presentation to the study center varied from 2 months to 31 years. Neonatal jaundice had been noted in 44.2%. Red cell transfusion requirement ranged from transfusion dependency [≥1 red cell unit/month; n = 13 (30.2%)] to a completely transfusion-free state [n = 14 (32.6%)]. The remaining 16 required occasional transfusions. Median hemoglobin was 7.8 g/dL (range, 2.8 to 15.3 g/dL). Supplemental Table S2 shows the detailed clinical and hematological presentation of enrolled patients. Heterogeneity was observed in erythrocyte morphology. Normocytic normochromic red cells were observed in eight cases (18.6%), of which five were not recently transfused. Acanthocytes were seen in most cases, with their percentage varying from a few to nearly 45% of the red cells. One case (HA53) had 25% to 30% stomatocytic red cells. In five cases, predominant stomatocytes were observed along with macrothrombocytopenia. Red cell morphology was not helpful in recently transfused patients. However, the persistent presence of stomatocytes alone or stomatocytes with macrothrombocytopenia in multiple samples corroborated the suspicion of stomatocytic disorders. Smear assessment was overall unhelpful in pinpointing the cause for hemolytic anemia once common etiologies were excluded. The commercial TruSight One Sequencing Panel, covering 4813 disease-causing genes, was initially applied in the 13 patients. Unexpectedly, NGS revealed potentially deleterious variants causing PK deficiency in five patients (HA72, HA82, HA97, HA111, and HA139) despite their previous screening test having shown normal PK enzyme activity. Of the five patients, two (HA72 and HA82) had homozygous PKLR variants (p.Asp331Gly and p.Gly332Ser), whereas three were compound heterozygotes. Compound heterozygosity for p.[Pro383Leu];[Arg486Trp] was found in HA97, p.[Arg488Gln];[Tyr564Ter] was found in HA111, and p.[Glu315Lys];[Arg486Trp] was found in HA139. Except for one patient (HA139) with p.[Glu315Lys];[Arg486Trp] variants, all others presented with neonatal jaundice and severe anemia and required regular blood transfusions. HA139 was a 40-year–old female with hemoglobin 7 to 8 g/dL and no hepatosplenomegaly who had required a transfusion during pregnancy. She had a corrected reticulocyte count of 4.2% and approximately 15% to 20% ovalocytes on the blood smear. Her sister, who was also anemic, with a corrected reticulocyte count of 40%, had been diagnosed as having hereditary ovalocytosis based on a blood film evaluation at the age of 15 years. The sister had subsequently undergone splenectomy at 28 years of age. Red cell morphology in the other cases was neither contributory nor misleading; most had normocytic normochromic cells with occasional acanthocytes and marked reticulocytosis. Uniformly high reticulocyte counts were not always observed in these PK-deficient patients. One patient (HA97), compound heterozygous for p.[Pro383Leu];[Arg486Trp], was initially suspected to have congenital dyserythropoietic anemia because of near-normal reticulocyte counts (on regular blood transfusions) and was enrolled for this study at 9 months of age because of one report of an uncorrected 17% reticulocytosis at 3 months of age. His sister, with a similar presentation, was also compound heterozygous for the same variants. Two patients (HA120 and HA129) had G6PD deficiency due to p.Arg417Cys (G6PD Guadalajara) and p.Tyr227Ser (novel, named as G6PD Chandigarh). Both presented with neonatal jaundice and had mild splenomegaly. HA129 with p.Tyr227Ser had required multiple blood transfusions in the neonatal phase, whereas the other patient (G6PD Guadalajara) had been transfused only once. Incubated osmotic fragility testing had revealed increased red cell fragility in both. No bite cells or Heinz bodies were seen, and the methylene blue reduction test was previously normal in both, likely on account of marked reticulocytosis. Glucose-6-phosphate isomerase (GPI) deficiency was found in two patients. A novel homozygous p.Phe304Leu variant (named as GPI Chandigarh) resulting in GPI deficiency was found in a male patient (HA101) who had required a blood transfusion only once. This variant was present in the 20–amino acid long α-helix (amino acids 290 to 309) in the protein.12Cordeiro A.T. Godoi P.H.C. Silva C.H.T.P. Garratt R.C. Oliva G. Thiemann O.H. Crystal structure of human phosphoglucose isomerase and analysis of the initial catalytic steps.Biochim Biophys Acta. 2003; 1645: 117-122Crossref PubMed Scopus (28) Google Scholar Clinically, he had mild anemia (hemoglobin, 10 g/dL) with normocytic normochromic red cells and 15% reticulocytes. Examination showed hepatosplenomegaly. His parents and asymptomatic siblings were heterozygous for the same variant. Another case, an infant (HA137), presented with neonatal jaundice and severe anemia, with 35% to 40% acanthocytic red cells and uncorrected reticulocyte count of 60%. She revealed a known homozygous pathogenic GPI variant p.Arg347His, and the case was previously reported.11Jamwal M. Aggarwal A. Das A. Maitra A. Sharma P. Krishnan S. Arora N. Bansal D. Das R. Next-generation sequencing unravels homozygous mutation in glucose-6-phosphate isomerase, GPIc.1040G>A (p.Arg347His) causing hemolysis in an Indian infant.Clin Chim Acta. 2017; 468: 81-84Crossref PubMed Scopus (15) Google Scholar This panel also revealed one case each of overhydrated hereditary stomatocytosis [OHS (RHAG)] and Mediterranean stomatocytosis/macrothrombocytopenia (ABCG5). The OHS patient had compensated hemolysis and a complex genotype for the RHAG gene.13Jamwal M. Aggarwal A. Sachdeva M.U.S. Sharma P. Malhotra P. Maitra A. Das R. Overhydrated stomatocytosis associated with a complex RHAG genotype including a novel de novo mutation.J Clin Pathol. 2018; 71: 648-652Crossref PubMed Scopus (5) Google Scholar All three affected siblings in a family with Mediterranean stomatocytosis/macrothrombocytopenia had a phenotype restricted to hematological manifestations along with short stature.14Jamwal M. Aggarwal A. Maitra A. Sharma P. Bansal D. Trehan A. Thapa B.R. Malhotra P. Das R. First report of Mediterranean stomatocytosis/macrothrombocytopenia in an Indian family: a diagnostic dilemma.Pathology. 2017; 49: 811-815Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar All the variants prioritized as potentially causal were predicted to be highly deleterious/harmful by in silico analysis by PolyPhen, SIFT, PROVEAN, MutPred, Mendelian Clinically Applicable Pathogenicity, and Combined Annotation Dependent Depletion. They were screened in 125 hematologically normal individuals and were found to be absent. Family members were screened by Sanger sequencing to establish causality and inheritance patterns. At the end of testing, all variants were classified as per American College of Medical Genetics and Genomics guidelines, 2015.15Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. Voelkerding K. Rehm H.L. ACMG Laboratory Quality Assurance CommitteeStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (14840) Google Scholar Variants and in silico findings are summarized in Table 1. Thus, the TruSight One Sequencing Panel was able to diagnose 84.6% (11/13) of the cases. Causative variants were not detected in two patients.Table 1NGS Findings Using TruSight One Sequencing Panel and in Silico AnalysisLaboratory IDGeneChrRead depth (X)GenotypeExonHGVScHGVSprs IDMAF (ExAC)SIFT ( 0.5)PROVEAN (>-2.5)MutPred (>0.75)M-CAP (>0.025)CADD (>20)HA120G6PDX26Hemi10/13NM_000402.3:c.1249C>Tp.Arg417Cys (G6PD Guadalajara)rs137852334NADel(0)PD (0.995)Del(-7.07)H(0.742)PP(0.928)26.9HA129G6PDX19Hemi6/13NM_000402.3:c.680A>Cp.Tyr227Ser (G6PD Chandigarh)NovelDel(0)PD(0.998)Del(-8.618)H(0.917)PP(0.911)27.7HA101GPI1971Homo11/18NM_001184722.1:c.912C>Gp.Phe304Leu (GPI Chandigarh)NovelDel(0)PD(0.864)Del(-5.655)H(0.706)PP(0.217)19.41HA137GPI1924Homo12/18NM_001184722.1:c.1073G>Ap.Arg358Hisrs1378535830.000008/1Del(0.01)PD(0.997)Del(-4.73)H(0.890)PP(0.280)31HA72PKLR119Homo7/11NM_000298.5:c.992A>Gp.Asp331GlyNovelDel(0)PD(0.997)Del(-6.555)H(0.951)PP(0.443)29.3HA82PKLR128Homo7/11NM_000298.5:c.994G>Ap.Gly332Serrs7736262540.00006/7Del(0)PD(1)Del(-5.500)H(0.940)PP(0.408)29.9HA97PKLR12613Comp het8/1110/11NM_000298.5:c.1148C>TNM_000298.5:c.1456C>Tp.Pro383Leup.Arg486TrpNArs1161006950.00004/30.0028/343Del(0)Del(0)PD(1)PD(0.996)Del(-8.027)Del(-5.987)H(0.909)H(0.635)PP(0.302)PP(0.187)27.933HA111PKLR11147Comp het10/1111/11NM_000298.5:c.1463G>ANM_000298.5:c.1692C>Gp.Arg488Glnp.Tyr564Terrs369183199Novel0.00002/2Del(0)PD(1)Del(-3.265)H(0.835)PP(0.185)3237HA139PKLR12113Comp het6/1110/11NM_000298.5:c.943G>ANM_000298.5:c.1456C>Tp.Glu315Lysp.Arg486Trprs1557959063rs1161006950.0028/343Del(0)Del(0)PD(1)PD(0.996)Del(-3.691)Del(-5.987)H(0.913)H(0.635)PP(0.465)PP(0.187)3233HA53RHAG6138Het3/10NM_000324.2:c.447T>Gp.Ile149Metrs1554174425NADel(0.01)PD(0.996)Neutral(-2.479)H(0.540)PP(0.021)14.24HA89ABCG5218Homo6/13NM_022436.2:c.727C>Tp.Arg243Terrs1194790660.00003/441HA102No causal variant foundHA116No causal variant foundWeb source for accession number (NM_) is National Center for Biotechnology Information Reference Sequence database (https://www.ncbi.nlm.nih.gov/RefSeq).CADD, Combined Annotation Dependent Depletion; Chr, chromosome; Comp het, compound heterozygous; Del, deleterious; ExAC, Exome Aggregation Consortium; G6PD, glucose-6-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; H, harmful; Hemi, hemizygous; Het, heterozygous; Homo, homozygous; HGVSc, Human Genome Variation Society (for coding) transcript nomenclature; HGVSp, Human Genome Variation Society protein nomenclature; ID, identifier; MAF, minor allele frequency; M-CAP, Mendelian Clinically Applicable Pathogenicity; NA, not available; NGS, next-generation sequencing; PD, probably damaging; PP, possibly pathogenic; PROVEAN, Protein Variation Effect Analyzer; SIFT, Sorting Intolerant from Tolerant. Open table in a new tab Web source for accession number (NM_) is National Center for Biotechnology Information Reference Sequence database (https://www.ncbi.nlm.nih.gov/RefSeq). CADD, Combined Annotation Dependent Depletion; Chr, chromosome; Comp het, compound heterozygous; Del, deleterious; ExAC, Exome Aggregation Consortium; G6PD, glucose-6-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; H, harmful; Hemi, hemizygous; Het, heterozygous; Homo, homozygous; HGVSc, Human Genome Variation Society (for coding) transcript nomenclature; HGVSp, Human Genome Variation Society protein nomenclature; ID, identifier; MAF, minor allele frequency; M-CAP, Mendelian Clinically Applicable Pathogenicity; NA, not available; NGS, next-generation sequencing; PD, probably damaging; PP, possibly pathogenic; PROVEAN, Protein Variation Effect Analyzer; SIFT, Sorting Intolerant from Tolerant. In the next phase of testing, 30 new subjects, as well as the two cases unsolved by TruSight One panel fulfilling the diagnostic criteria, were subjected to targeted resequencing, this time using a highly customized panel of 55 genes implicated in inherited anemias. Wide genetic heterogeneity was observed. Three patients (HA39, HA165, and HA178) had potentially deleterious variants causing PK deficiency. Homozygous p.Arg479His was found in one (HA39), whereas two had compound heterozygous variants: c.[1618+1G>C];p.[Gly332Ser] in HA165 and p.[Ala66Gly];[Arg486Trp] in HA178. PK enzyme activities had been normal in all cases, with PKLR defects on NGS, likely attributable to reticulocytosis or recent transfusions. Two patients had Mediterranean stomatocytosis with macrothrombocytopenia, harboring a probable causal homozygous splice region variant in ABCG5 (c.1464-1G>A) and a homozygous nonsense variant in ABCG8 (p.Ser129Ter) genes, respectively (HA195 and HA142). HA195 also had coinherited G6PD deficiency (G6PD Kerala-Kalyan) with Mediterranean stomatocytosis/macrothrombocytopenia (ABCG5). His sister too had a similar phenotype and ABCG5 genotype. HA142 was heterozygous for -α3.7-kb deletion in
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